skin irritation test for the prediction of acute ......a total of 20 test substances, consisting of...

Version 5.0 In vitro Skin Irritation TestUsing the Reconstructed Human Epidermis (RHE Sterlab) model Sterlab

Laboratories sur 32 Standard Operating Procedure

SKIN IRRITATION TEST FOR THE PREDICTIONOF ACUTE SKIN IRRITATION OF CHEMICALS

WITH STERLAB’S RHE:42 MINUTES APPLICATION + 42 HOURS POST-INCUBATION

Standard Operating Procedure (SOP)

Siège social : STERLAB - 2720 chemin Saint Bernard – 06224 Vallauris CEDEX – FranceTél : +33 (0)4 97 24 58 58 – Fax : +33(0)4 97 24 58 59 Email : [email protected] – Web : www.sterlab.com

S.A.S AU CAPITAL DE 700000 € - RCS ANTIBES 487 829 483 – SIRET 487 829 483 00014 – NAF 7219Z

mailto:[email protected]

http://www.sterlab.com/



1. Protocol IntroductionSterlab Reconstructed Human Epidermis (RHE) are used for the classification of chemicals concerningtheir skin irritating properties by measurement of its cytotoxic effect, as reflected in the MTT assay.

OBJECTIVES & APPLICATIONS

Type of Testing:Replacement

Level of Toxicity Assessment:Toxic potential, toxic potency, hazard identification

Purpose of Testing:Prediction of skin irritation potential

Context of Use:In vitro skin irritation tests are regulatory accepted.After the update of the ECVAM performance standards in 2009 the OECD guideline for the testing ofchemicals, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method 439” was adopted.

Applicability Domain:This test is designed for chemicals. The protocol was established for liquids, viscous and solid testsubstances.

BASIS OF THE METHOD

Dermal irritation is induced by chemicals which penetrate the stratum corneum and lead to damagesand cell loss of the underlying cells layers.The relative viability of the treated tissues is measured at the end of the treatment exposure (42minutes) followed by a post-exposure period (42 hours) using a cell viability test.A cutoff value of 50% viability of the negative control value was used to classify test substances asirritant (I) or non classified as irritant (NI).







ABBREVIATIONS & DEFINITIONS

I: IrritantID: identityMDS: Methods Documentation SheetMTT: 3-[4, 5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromideNC: Negative ControlNI: Non IrritantOD: Optical DensityPBS: Phosphate Buffer SalinePC: Positive ControlQC ID: Quality Control IdentityRHE: Reconstructed Human EpidermisRT: Room TemperatureSDS: Sodium Dodecyl SulfateTriton X100: Triton

EXPERIMENTAL DESCRIPTION

Endpoint & Endpoint detection:Cell viability is measured by dehydrogenase conversion of MTT, present in cell mitochondria, into ablue formazan salt that is quantitatively measured after extraction from tissues (Mosmann T., 1983).The reduction of viability of tissues exposed to chemicals in comparison to negative controls is used topredict skin irritation potency.

Endpoint Value:The quantitative cell viability is determined as a percentage of the negative control.

Test System:The Sterlab RHE consists of normal human-derived keratinocytes cultured for 17 days at the air-liquidinterface. The RHE model is cultured using a chemically defined growth medium. On day 17, a highlydifferentiated and stratified epidermis model is obtained comprising the basal, spinous and granularlayers, and a multilayered stratum corneum. The RHE model presents a histological morphologycomparable to the in vivo human tissue.A generic description of general and functional conditions that skin models need to comply with canbe found in the OECD Test Guideline 439.The Sterlab RHE are cultured on inserts 0.5 cm2 polycarbonate filters and shipped world-wide onagarose together with Sterlab Maintenance Medium.The quality system of the Sterlab Laboratories is ISO 9001:2008. Each batch production is providedwith quality controls values and recommendations such as storage conditions, RHE instructions foruse, batch number and origin, histology, cell viability (MTT OD > 0.8), barrier function integrityperformed at day 17 (4.0 ≤ ET50 (hr) ≤ 9.0), absence of bacteria, fungi, HIV, and Hepatitis B, C.







Basic Procedure:On day of receipt, Sterlab RHE are conditioned by incubation at 37°C for release of transport-stressrelated compounds and debris at least 2 hours or overnight.Each test substance (test chemicals, negative and positive controls) is topically applied concurrently onthree tissues replicates for 42 minutes at room temperature (RT comprised between 18°C to 24°C).Exposure to the test substance is followed by rinsing with phosphate buffer saline (PBS) andmechanically dried. RHE are then transferred to fresh medium and incubated at 37°C for 42 additionalhours. Cell viability is assessed by incubating the tissues for 3 hours with 0.3 mL MTT solution (0.5mg/mL). The formazan crystals are extracted using 1.5 mL isopropanol for 2 hours at RT and quantifiedby spectrophotometry at 550 ± 30 nm wavelength. Sodium Dodecyl Sulphate (SDS 5%), and PBS are used as positive and negative controls, respectively.For each treated tissue, the cell viability is expressed as the percentage of the mean negative controltissues.

Data analysisIrritation potential of test substance is determined according to the EU classification (R38 or no label).The mean relative tissue cell viability above 50 % predicts its non-irritancy potential. The predictionmodel is defined as described below:

In vitro results In vivo classification

Mean tissue cell viability >50% No Category (No Cat.)

Mean tissue cell viability ≤50% Category 2 (Cat. 2)

Test compounds and Result summaryA total of 20 test substances, consisting of 10 in vivo irritants and 10 in vivo non classified as irritantwere proposed in the Performance Standards for applying human skin models to in vitro skin irritationtesting, in the ECVAM Skin Irritation Validation Study (ECVAM SIVS, 2007).The blind catch up validation study was performed under GLP like conditions in the three participatinglaboratories, good reproducibility was achieved in these three laboratories.Correct predictions of the skin potential for those twenty test substances were assessed with 100 % sensitivity, 70 % specificity (MTT only) and 85 % accuracy (see Table).

LaboratorySpecificity [%] Sensitivity [%]

All runs All runs

Lab 1 70 100

Lab 2 70 100

Lab 3 70 100

Table: Summary of the predictive capacity (specificity and sensitivity) in the three laboratoriesconsidering either all chemical runs or only those chemicals, which had three valid runs.







METHOD

Modification of the methodNon

DiscussionIn contrast to the use of laboratory animals or excised human skin this method offers a highreproducibility due to standardized materials and processes during the production. Furthermore theSterlab RHE is based on human cells to predict effects on humans and serves as a completereplacement of the in vivo acute skin irritation test in rabbits.

Known laboratory useSterlab RHE is used in industry and academia for research, efficacy and toxicology testing.

Proprietary and confidential issuesThe Reconstructed Human Epidermis technology associated production methods and media areproprietary to Sterlab, France. No intellectual property rights are associated with the present test method.







2. Technical DescriptionVersion 1.0, June 2014

STERLAB RHE IN OECD TEST GUIDELINE 439

Contact Person: Dr. Anna GINOSIAN, PhDFull Address: Sterlab Laboratories2720 chemin Saint Bernard06220 VALLAURIS, FranceTel: +33 (0)4 97 24 58 52Fax: +33 (0)4 97 24 58 59E-mail: [email protected]

CELL / TEST SYSTEM

Health and Safety issuesSterlab tissues are manufactured using epidermal cells taken from healthy volunteer donors negativeto anti-HIV-1 and 2, to hepatitis C antibodies, to hepatitis B antigens.Nevertheless, normal handling procedures for biological materials should be followed:

(a) Wear gloves during handling with the skin and kit components;(b) After use, the epidermis, the material and all media in contact with it, should be

decontaminated (e.g. using a 10% solution of bleach or appropriate containers).(c) Examine all kit components for integrity.

Please contact Sterlab Laboratories, if you have any questions or concerns.

Test system description:The three-dimensional Reconstructed Human Epidermis (RHE) model, commercialized by Sterlab(Vallauris, France) consists of normal human keratinocytes cultured for 17-days on an insert 0.5 cm 2

polycarbonate filter at the air-liquid interface. The RHE model is cultured using a chemically definedgrowth medium.

Quality control:The RHE models are manufactured according to defined ISO9001v2008 quality assurance procedures.All the RHE models are free of viruses, bacteria and mycoplasma. The quality of the final RHE productis assessed by a MTT cytotoxicity test of untreated tissues, barrier function integrity with Triton X1001% (4.0 ≤ ET50 (hr) ≤ 9.0) and by histological examination.








TISSUES AND MEDIA

RHE set and media are provided by Sterlab.

The Reconstructed Human Epidermis (205EPID05) and the necessary culture media (Maintenancemedium MILM, and Growth medium MILC) are usually shipped on Monday. They are received one ortwo day(s) following the shipment (for Europe). Results of the quality controls are supplied with thesets. A batch production is delivered only if quality controls criteria correspond to a normal histology(absence of significative alterations), cell viability (MTT OD > 0.8), barrier function integrity with TritonX100 1% (4.0 ≤ ET50 (hr) ≤ 9.0) and absence of bacteria, fungi, HIV, and Hepatitis B, C.

Expiration and storage

Reference Description Storage condition Shelf life

205EPID05 RHE, small size (0.5 cm²), day 17 37 ± 0.5°C / 5% CO2 10 days

205MILM125 Maintenance medium 2 - 8°C 10 days

205MILC125 Growth medium 2 - 8°C 10 days

Receipt of materials supplied by SterlabExamine all kit components for integrity. If there is a concern call Sterlab.Document MDS: RHE set - Sterlab materials receipt (Annex 1).

EQUIPMENT

Consumables:

Extra 6-well plate - sterile To transfer tissue inserts to fresh medium

24-well plate - sterile For the 42 min application + MTT incubation +formazan extraction steps

96-well plate - sterile For reading formazan extract

Sterile absorbent paper / sterile gauze To remove agarose fragments or to dry inserts

Circular nylon meshes Ø = 7.5mm Use as a spreading aid for liquid test materials

Adhesive tape or Parafilm M Covering plates during formazan extraction

Tips For adjustable pipette







Fixed Equipment:

Laminar flow hood For safe work under sterile conditions

Non sterile ventilated cabinet or laminar flow hood with chemical filter

For safe work with chemicals, applications, washes

Cell incubator (37°C, 5% CO2, 95% relative humidity) For incubating tissues prior to and during assays

Laboratory balance (accuracy 0.1mg) For pipette verification and test substance weighing

96-well plate spectrophotometer (550 ± 30 nm) For reading OD

Plate shaker For extraction of formazan

Timers To be used during application of test materials

Sterile forceps For handling tissue inserts

Plastic wash bottles For collecting PBS rinses

1 L beaker For rinsing tissues with PBS

Small glass weight boats For weighing powders

1 gauged flask For SDS 5% solution preparation

Mortar and Pestle For grinding granular solids

Adjustable Pipette / multi-step Pipette For pipetting 1 mL assay medium

Adjustable Pipette / multi-step Pipette For pipetting 300 μL MTT / medium

Adjustable Pipette For pipetting 750 μL propan-2-ol

Adjustable Pipette For pipetting 200 μL formazan extract from 24-well plate into 96-well plate

Positive displacement pipette for 30μL For application of liquid and viscous test materials

Multi-pipette + adapter for 25mL tip For washing

Vacuum source/trap For aspirating media and solutions

Equipment verificationIt is strongly recommended to use regularly verified equipment. Maximum time interval between twoverifications is specified for each apparatus necessary in this protocol in Methods DocumentationSheet, MDS: Main equipment verification (Annex 2).If the last verification date does not fit the requested specifications, proceed to verification beforetesting and record it in MDS: Main equipment verification and MDS: Detailed equipment verification(Annexes 2 and 3).







OTHER MEDIA AND REAGENTS

Dulbeccos’ PBS without Ca2+ and Mg2+

(Biowest Absyc, L0615) Use as negative control, MTT diluent, and for rinsing tissues

MTT – Thiazolyl Blue Tetrazolium Bromide (Sigma, M5655, cell culture tested, purity min. 97.5%)

For the MTT assay

5 % (aq) SDS [CAS N°151-21-3] (Sigma,L4509, purity min. 98.5%)

To be used as positive control

Sterile H2O (distilled or aqua pure) For powder applications

Isopropanol [CAS N°67-63-0] (Sigma, 190764)

For extraction of the formazan crystals

MTT solution preparation

Safety precautions

MTT (R26 R22 R36 R37 R38) - MTT solution is light sensitive. Protect it from light using silver paper or appropriate material.

Isopropanol (R11 R36 R67 S7 S16 S24/25 S26)

Work in ventilated cabinets: to prevent accidental contact wear protective gloves, and if necessary amask and/or safety glasses.

1) MTT stock solution preparationDissolve MTT powder (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-AldrichM-5655) to a final concentration of 5 mg/mL in PBS. Always protect the solution from light. Thesolution is filtered with 0.22 μm filtration. Discard the MTT solution in 1 mL aliquots in sterile dark 1.5mL microtubes. Storage: 1 year at –20°C.Document MDS: MTT stock solution (Annex 4).

2) MTT ready to use solution preparation (day of testing)On day of testing, thaw the MTT stock solution (5 mg/mL) and dilute it with pre-warmed maintenancemedium at room temperature up to 0.5 mg/mL.







TEST SUBSTANCES

Safety instructions

Store test substances in ventilated safety cup boards. Respect special store conditions if necessary (special temperature, protected from light, etc.).

Non-coded test substances should be handled following material safety datasheet.

Unknown and coded test substances with no or incomplete safety handling information shouldbe considered as irritating and toxic and must be handled with maximum care. In accordance with test substance safety guidelines: use safety ventilated cabinet, wear gloves, eye and face protections.

Test substances identificationMain information concerning the test substances (name or code, total weight, reception date,expiration date, physical consistence, stocking conditions) should be registered in MDS: Testsubstances (Annex 5).

Negative control solutionPBS is used as negative control.Document MDS: Solutions preparation (Annex 4).

Positive control solutionPrepare extemporaneously SDS 5% solution (Positive Control). Storage: 1 month at 2 - 8C°. Document MDS: Solutions preparation (Annex 4).

Note: The % SDS solution must be made in weight / volume (weighing of the SDS then add distilled water untilthe necessary volume to reach the final concentration of 5 % W/V) e.g. 1g of pure SDS qsp 20mL water using agauged flask.







3. METHOD

Before starting the 3 independant runs, verifications should be done with the 20 chemicals (ideally theweek before the study).

First the coloration of each substance has to be identified : document MDS : Test substanceidentification (Annex 5). Thus an additional condition will be performed with the coloring testsubstances (named condition 2 in the Flowchart page 12)

Moreover, relative conversion of MTT by the tissue being the parameter evaluated in this test method,it is therefore necessary to assess the non specific reduction of MTT by the test substance used: alltest substances should be put in contact with MTT solution as described below.

3.1 Determination of the non specific reduction of MTT by test substance

3.1.1 Check-method for possible direct MTT reduction with test substances

1. Fill wells of 24-well plate with 300μL of MTT ready to use solution (0.5 mg/mL)2. Add 16μL or 16 mg of the test substance or water for control. Mix.3. Protect from light and incubate the mixture for 3 hours ± 5 min at 37 ± 0.5°C.4. Proceed to visual scoring of MTT interaction as follow:

Negative control (water): yellow Test substances which do not interact with MTT: yellow Test substances interacting slightly with MTT: light blue Test substances interacting strongly with MTT: dark blue

5. Document MDS: Test substance identification (Annex 5).

If the MTT solution color becomes blue or purple, the test substance interacts with the MTT.It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction ofthe MTT (i.e. by using killed epidermis).For each MTT-interacting test substance previously detected, and in addition to the normal procedure,3 killed test substance treated tissues are used for the MTT evaluation following the same protocol asfor living tissues (named condition 3 in the flowchart page 12). Three killed untreated tissues are usedas negative controls (untreated killed tissues may exhibit little residual NADH and dehydrogenaseassociated activity).







3.1.2 Killed epidermis for MTT-interaction substances (if necessary)

1. Transfer living epidermis in 24-well plate and place them at -20°C or -80°C for at least 24 hoursbefore test (3 tissues / MTT-interacting test substance).

2. Thaw killed tissues, before use, on 300μL of maintenance medium for 1 hour ± 5 min at roomtemperature.

3. Further use of killed tissues is similar to living tissues (see 3.3).4. Document MDS: Killed tissues for MTT-interacting test substances (Annex 6).

3.2 Guidance FLOWCHART for adapted controls choice based on test substancescoloring and/or direct MTT reduction potency.

CONDITION 1 CONDITION 2 CONDITION 3

All test substances(standard, MTT interacting,

coloring test substances)Coloring test substances

MTT-interacting testsubstances

Use of living RHE tissues+ PBS Negative control tissues+ SDS Positive control tissues

Use of living RHE tissuesUse of killed RHE tissues

+ Untreated Negative controlkilled tissues

42 min exposure + 42 hours post-incubation

3 hoursMTT incubation

3 hoursMaintenance Medium

incubation

3 hoursMTT incubation

2 hours isopropanol extraction

OD = Specific OD + Non-SpecificOD

OD = Non-Specific Staining(NSS)

OD = Non-Specific MTTreduction (NSMTT)







3.3 Routine Protocol

3.3.1 Pre-incubation stepThis step should be conducted under sterile conditions.Before starting pre-incubation step, the maintenance and growth media should be pre-warmed onlyat room temperature (and not at 37°C).

Pre-incubation step for tissues receipt at day 18 (usually on Tuesday)1. Fill an appropriate numbers of 6-well plates with 1 mL growth culture medium (MILC).2. Remove the adhesive tape from the agarose plate containing epidermal tissues and open the

24-well plates. See Picture 1.3. Use sterile forceps to take off tissues from the agarose, clean the bottom of the insert on

sterile absorbent paper or gauze to remove eventual remaining agarose pieces.4. Check visually that no agarose is remaining and transfer the tissue on fresh medium by first

slopping the insert before complete insert setting. See Picture 2.5. Place the RHE tissues at 37°C, 5% CO2 until test substance application.

Pre-incubation step for tissues receipt at day 19 (Wednesday)1. Proceed to pre-incubation step for at least 2 hours.2. Fill an appropriate numbers of 24-well plates with 300 μL growth culture medium (MILC).3. Remove the adhesive tape from the agarose plate containing epidermal tissues and open the

24-well plates. See Picture 1.4. Use sterile forceps to take off tissues from the agarose, clean the bottom of the insert on

sterile absorbent paper to remove eventual remaining agarose pieces.5. Check visually that no agarose is remaining and transfer the tissue on fresh medium by first

slopping the insert before complete insert setting at the air-liquid interface.See Picture 2.

6. Place tissues at 37°C, 5% CO2 until test substance application.

Picture 1 Picture 2







Application of test substances and rinsing on day 19 (Wednesday)

Safety precautions

Irritant materials are dangerous: It is thus recommended to work in laminar flow hood with chemical filter or in ventilated cabinets and wear gloves, coat, as necessary.

3.3.2 Plates preparation

Application plates1. Pre-warm the maintenance culture medium at room temperature.2. Label a 24-well plate by condition: 3 wells per test substance (code number, 3 replicates),

positive control (PC), negative control (NC), substances for killed tissues (code number, 3replicates) and coloring test substances (code number, 3 replicates) respectively.

3. Fill the 3 wells with 300 μL pre-warmed maintenance culture medium.4. Use sterile forceps to transfer tissues by first slopping the insert before complete insert setting

at the air-liquid interface.

Post-incubation plates1. Pre-warm the growth culture medium at room temperature.2. Label a 6-well plate by condition: 3 wells per test substance (code number, 3 replicates),

positive control (PC), negative control (NC), substances for killed tissues (code number, 3replicates) and coloring test substance (code number, 3 replicates) respectively.

Fill the 3 wells with 2 mL pre-warmed growth culture medium.

3.3.3 Topical applications: 42 minutes treatmentIt is strongly recommended to perform this step under sterile conditions.Three tissues per test substance should be used (3 replicates). The application order is important sinceit will be the same for washing.Note: Keep 1 minute interval between each tissue application. We recommend keeping some minuteswithout test substance application just before washing in order to be ready in time for this latter.Record the exact timings and document the correspondent MDS: Application timing (Annex 9).Due to the application timing of 42 minutes, the application and rinsing phases should be performedin minimum two steps for testing the internal controls (NC and PC), the 20 test substances, thecoloring test substances and the substances for killed tissues.

Liquid and viscous test substances1. Dispense 16 μL ± 0.5 μL (i.e. 32 μL/cm²) of the undiluted test substance on the top of each

epidermis tissue (living tissue and killed tissue if necessary): 3 per test substance, using







positive displacement pipette. Use the tip to spread the test substance gently on the epidermistopical surface. See picture 3.

2. Carefully apply a nylon mesh (Ø = 7mm) on the whole surface with forceps.See pictures 4 and 5.

3. Document MDS: Killed tissues for interacting test substances (annex 6), MDS: Additionalcoloring test substances (annex 7) and MDS: weighing of solid and sticky test substances(annex 8)

Picture 3 Picture 4 Picture 5

Solid tests substances1. If necessary, the test substance should be crushed to a fine powder using a mortar and a

pestle.2. Gently spread 10μL of distilled water using a positive displacement pipette to the epidermal

surface in order to improve further contact between the powder and the epidermis.3. Use special glass weigh boats to apply 16 mg ± 2 mg (i.e. 32 mg/cm²) of the powder to the

epidermis surface. If necessary, spread it on the epidermal surface. 4. Document MDS: Killed tissues for interacting test substances (annex 6), MDS: Additional

coloring test substances (annex 7) and MDS: weighing of solid and sticky test substances(annex 8)

See Pictures 6 and 7.







Picture 6 Picture 7

Sticky test substances1. Allow for the tare with nylon mesh and directly weigh 16 mg ± 2 mg (i.e. 32 mg/cm²) and

spread sticky test substance on this latter.2. Apply the test substance coated side of the nylon mesh on the epidermal surface and spread it

gently on the whole surface.3. Document MDS: Killed tissues for interacting test substances (annex 6), MDS: Additional

coloring test substances (annex 7) and MDS: weighing of solid and sticky test substances(annex 8)

Three tissues per test substance should be used. The application order is important since it will be thesame for rinsing.

Keep the plate (lids on) containing the treated RHE tissues for 42 minutes exposure (± 1 min) in theventilated cabinet sterile conditions at room temperature.

3.3.4 Rinsing and drying stepsIt is strongly recommended to work in laminar flow hood to prevent contamination.End of the treatment and removal of the test substance after 42 minutes (± 1 min) exposure at roomtemperature.Strictly respect the application order (time based recorded in Annex 9).In order to prevent pollution the lids should be put on the plates continuously during the rinsing anddrying steps. We recommend also to put a lid on the sterile PBS container.

Liquid, viscous, sticky and solid test substances1. Fill a multi-pipette (adjusted for a 1 mL distribution) with 20 mL sterile PBS2. Open the 24-well plate.3. Remove the nylon mesh with fine forceps from the epidermal surface of a treated tissue.4. Take the treated tissue with sterile forceps and close the 24-well plate (to protect the other

tissues from washing solution projections). See Picture 8.







Picture 8

Rinsing step for treated tissues1. Place a funnel in a large beaker (to avoid underneath projections/contaminations of the RHE

tissues).2. Maintain the insert over the large funnel and rinse thoroughly 20 times with 1mL PBS at a 5-8

cm distance from the insert to remove all residual test substance from the epidermal surface.3. After the last rinsing, empty the insert at the most (for example, knock the forceps on

the beaker, insert turned upside down).4. Dry the insert bottom on a sterile absorbent paper or gauze for 1-2 seconds.5. Sweep gently the surface of the stratum corneum with both ends of a cotton tip (5-6 turns per

end). See Picture 9.6. Transfer the washed tissue on 2 mL growth culture medium (6-well plate designed as post-

incubation plates, see 3.3.2) by first sloping the insert before complete insert setting.7. Document MDS: Rinsing timing (Annex 9).

Picture 9







3.3.5 Post treatment incubation: 42 hoursIncubate the treated, rinsed and dried epidermis tissues at 37°C, 5% CO2, 95% humidified atmospherefor 42 hours (± 60 min) in the post-incubation plates pre-warm with growth culture medium.Incubation start time corresponds to last tissue rinsing time of each set.See MDS: post-incubation timing (Annex 9).

3.3.6 MTT testTissue viability is assessed by MTT reduction measurement, after the 42 hours (± 60 min) incubation at37°C, 5% CO2, 95% humidified atmosphere.

Incubation in MTT solution1. Prepare MTT ready to use solution according to page 9. 2. Label an appropriate numbers of 24-well plates3. Fill the 24-well plates with 300 μL MTT from light by wrapping in aluminum paper until tissues

transfer in MTT. Sweep excess culture medium on the unit bottom of the tissue with absorbentpaper.

4. Transfer the treated tissues in the pre-filled MTT 24-well plates, by first slopping the insertbefore complete insert setting at the air-liquid interface. Respect the application order.

5. Incubate for 3 hours (+/- 5 min) at 37°C, 5% CO2, 95% humidified atmosphere.6. Document MDS: MTT incubation (Annex 9).

Incubation in Maintenance Medium for 3 hoursColoring test substance controls must follow a similar treatment to MTT assay but avoiding contact toMTT. Corresponding tissues will thus be incubated in Maintenance Medium.

1. Label an appropriate numbers of 24-well plates.2. Fill the 24-well plates with 300 μL Maintenance Medium.3. At the end of the 42 hours post-incubation period, sweep excess culture medium on the unit

bottom of the tissue with absorbent paper and transfer dye test substance control tissues inthe pre-filled 24-well plates, by first sloping the insert before complete insert setting at theair-liquid interface.

4. Check the absence of air bubbles.5. Incubate for 3 hours ± 5min at 37°C, 5% CO2, 95% humidified atmosphere.6. Document MDS: Start of 3 hours incubation time (Annex 9).







Formazan extraction7. Label an appropriate numbers of new 24-well plates similarly to those labeled for the previous

step.8. Fill the plate(s) with 800 μL isopropanol (undiluted) at the end of the 3 hours ± 5 min

incubation in MTT solution (Annex 9). Use forceps to transfer treated tissues.9. Dry the insert bottom of the treated tissue on absorbent paper.10. Transfer the tissues in isopropanol solution.11. Add 700 μL isopropanol solution on the top of each tissue.12. Ensure that tissue is completely covered by the isopropanol solution.13. Consciously protect plate(s) from evaporation by stretching Parafilm over the plate and adding

the lid on the plate.14. Incubate for 2 hours ± 5 min at room temperature for formazan extraction.15. Document MDS: Start of isopropanol extraction time (Annex 9).

Absorbance / optical density measurements16. At the end of the formazan extraction incubation time, open the plate.17. Remove the Parafilm.18. Document in MDS: OD reading (Annex 9).19. Isopropanol solution is used as blank (6 replicates)20. Maintain the insert with forceps.21. Pierce tissue and polycarbonate filter with a tip in order to get the whole extraction solution in

the corresponding well.22. Homogenize the extraction solution by pipetting 3 times up and down to complete formazan

crystals solubilization.23. Transfer 3 x 200 μL extraction solution per well into a 96-well labeled plate.24. Read the Optical Densities (OD) using a 96-well plate spectrophotometer: the concentration of

formazan is measured by determining the OD at 550 ± 30 nm.25. All data generated by the 96-well plate spectrophotometer should be printed after each

reading and considered as raw data.26. Identify ODs with conditions and tissues (replicate) studied on the raw data documents.27. Perform the Quality Control of the raw data.







4. ACCEPTANCE CRITERIANegative control (NC) acceptance criteria: The NC data meet the acceptance criteria if the mean ODvalue of the 3 tissues is ≥ 0.8 at 550 ± 30 nm according to the historical database.

The Standard Deviation value is considered as valid if it is ≤ 18%, according to the PerformanceStandards (ECVAM SIVS, 2007). The SD value is calculated over replicates for a given run.

Positive control (PC) acceptance criteria: The PC data meet the acceptance criteria if the meanviability, expressed as % of the NC, is < 40% and the Standard Deviation value is ≤ 18%.

Batch acceptance criteria: All test substance data from one batch are considered as valid if both thenegative and the positive controls data fulfill the above criteria requirements.

Test substance data acceptance criteria: The inter-batch mean viability is calculated from the threeindependent assays or runs using intra-batch tissue mean (3 replicates / tissue and 3 tissues per run).Standard deviation of each intra-batch mean should not be > 18%. For a given test substance, if onebatch predicts a different class of irritation, the test substance must be retested in one additionalbatch (4th run). In this case, results from all four batches (4 runs) will be considered for the final meanand analyzed, except if technical problems are identified for the batch.

5. DATA ANALYSIS / CALCULATION OF RESULTS5.1 Data Calculation Step

Blank Calculate the OD mean from the 6 replicates for each plate: ODblank

Use the blank value to calculate corrected value for substances contained in the same 96-wellplate

Negative PBS-treated controls Calculate the OD mean per tissue (3 replicates) ODNC

The mean OD for all tissues corresponds to 100% viability = mean ODNC

Positive SDS-treated control Calculate the OD mean per tissue (3 replicates) ODPC

Calculate the viability per tissue %PC = [ODPC / mean ODNC] x 100

Tested compound Calculate the OD mean per tissue (3 replicates) ODTT

Calculate the viability per tissue %TT = [ODTT / mean ODNC] x 100

For each test substance (including PC) mean viability and standard deviations are calculated.







5.2 Data calculations for MTT-interacting substancesTest substances that interfere with MTT can produce non-specific reduction of the MTT. It is necessaryto evaluate the OD due to non-specific reduction and to subtract it before calculations of viability %.

Non-specific MTT reduction calculation (NSMTT) ODKu: untreated killed tissues OD ODKT: test substance treated killed tissues OD NSMTT = [(ODKT- ODKU) / mean ODNC] x 100

If NSMTT is > 30% relative to the negative control: additional steps must be undertaken if possible orthe test substance must be considered as incompatible with the test.

Treated tissue True MTT metabolic conversion (TODTT) ODTT: test substance treated viable tissues TODTT = [ODTT – (ODKT – ODKU)] Relative viability % = [TODTT / mean ODNC] x 100

5.3 Data calculations for coloring test substances able to stain tissues

For chemicals detected as able to color the tissues, it is necessary to evaluate the non-specific OD dueto the residual chemical staining (unrelated to any mitochondrial activity) and to subtract it beforecalculations of the “true” viability %.

Non-specific Staining % (NSS %) ODCT: coloring test substance treated tissue (incubated in Maintenance Medium before

isopropanol extraction) ODPBS: control PBS treated tissue (incubated in Maintenance Medium before isopropanol

extraction) NSS%= [ODCT / ODNC] x 100

If NSS % is > 30% relative to the negative control: additional steps must be undertaken if possible orthe test substance must be considered as incompatible with the test.

True MTT metabolic conversion (TODCT) ODCT: coloring test substance-treated tissues (MTT incubation) TODCT: true MTT metabolic conversion for coloring test substance treated tissue. TODCT = ODTT – ODCT

Relative viability % = [TODCT / mean ODNC] x 100







5.4 Data calculations for coloring test substances which are also MTT-interacting testsubstancesCalculate corresponding NSMTT and NSS.If (NSMTT % + NSS %) is > 30% relative to the negative control: additional steps must be undertaken ifpossible or the test substance must be considered as incompatible with the test.

True MTT metabolic conversion for dye test substances which are also MTT-interacting test substances (TODDTT)

ODCT: coloring test substance-treated tissues (MTT incubation) TODCT: true MTT metabolic conversion for coloring test substance treated tissue. TODCTT = [ODTT – (TODCT+ TODTT)] Relative viability % = [TODCTT / ODNC] x 100

6. PREDICTION MODELAccording to EU classification, the irritancy potential of test substances is predicted for distinguishingbetween R38 skin irritating and no-label (non-skin irritating) test substances OECD TG 404 & MethodB.4 of Annex V to Directive 67/548/EEC. In the present study, the irritancy potential of test substancesis predicted by mean tissue viability of tissues exposed to the test substance. The test substance isconsidered to be irritant to skin (R38), if the mean relative viability after 42 minutes exposure and 42hours post incubation is less or equal (≤) to 50% of the negative control. The prediction model (PM) isdescribed below:

In vitro results In vivo classification

Mean tissue cell viability >50% No Category (No Cat.)

Mean tissue cell viability ≤50% Category 2 (Cat. 2)

Document all remarks, comments on protocol modifications in Annex 13.







7. ANNEXES

Annex 1 - Methods Documentation Sheet:

RHE SET - STERLAB MATERIALS RECEIPT

Laboratory: ……………….. Study N°: ……………….. Assay N°: ……………..

STERLAB set receipt date: ……………………

Quantity Batch N° Expiration date

Sterlab RHE ………………… ……………………… ………………………

Maintenance culture medium ………………… ……………………… ………………………

Growth culture medium ………………… ……………………… ………………………

Remarks: ………………………………………………………………………………………………………………………………......

……………………………………………………………………………………………………………………………………………………..

……………………………………………………………………………………………………………………………………………………..

ID: ………………….. Date: ……………….. Signature: ……………

QC ID: …………….. Date: ……………….. Signature: …………….








EQUIPMENT VERIFICATION


EquipmentMaximum time

interval between twoverifications

Last verificationDate

Verification week of testing ifnecessary ()

Laminar flow hood 6 months

Non –sterileventilated Cabinet 2 years

Laminar flow hoodwith chemical filter 2 years

Incubator 3 months

Refrigerator 3 months

Freezer (-20°C) 1 year

Pipettes 6 months

Spectrophotometer 1 year

Balance 1 year










DETAILED EQUIPMENT VERIFICATION


Identification

Laminar flow hood: ……………….. Non–sterile ventilated cabinet: ……………….. Laminar flow hood with chemical filter: ………………..

Incubator verificationIncubator N° CO2 5 ± 0.5% Temperature 36.5 ± 1°C Water bath level ()


Balance verificationN°: ……………….. 10 mg weight 1 g weight

Weighing 1 ……………….. mg ……………….. gWeighing 2 ……………….. mg ……………….. gWeighing 3 ……………….. mg ……………….. g

Mean ……………….. mg ……………….. gTolerance 10 ± 0.1 mg 1000 ± 0.5 mg


Pipettes verificationBalance

N°:…………..PipetteN°……….

PipetteN°……….

PipetteN°……….

Volume ……….μL ..…….μL ...…….μLWeighing (mg) 1Weighing (mg) 2Weighing (mg) 3

MeanSD

CV (%)Tolerance 5% 5% 5%










SOLUTIONS PREPARATION


Positive control: SDS 5% solution in distilled water (w/v)

Reference Batch N°: Expirationdate Weight Vehicle

volumeSDS solutionin distilled

water…………………… …………………… …………………… …………………… ……………………

MTT solutionin PBS

…………………… …………………… …………………… …………………… ……………………










TEST SUBSTANCES IDENTIFICATION


MTT interaction Start of incubation time : ……………… End of incubation time : ………………

Test substanceName or code

StorageTemperature

PhysicalConsistence* Colour

MTT InteractionBlue colorYes / No

Remarks

* Physical consistence: L = Liquid; V = Viscous ; S = Solid ; St = Sticky










KILLED TISSUES FOR MTT-INTERACTING TEST SUBSTANCES


Test substanceName or Code

N°……….. N°……….. N°……….. N°……….. N°……….. N°……….. N°………..

Solids Weightbefore application

(mg)W1

Tissue 1

Tissue 2

Tissue 3

Test substanceweight (mg)

W2

Tissue 1

Tissue 2

Tissue 3

Solids weight afterapplication (mg)

W3

Tissue 1

Tissue 2

Tissue 3

Test substanceweight

applied (mg)W=(W1+W2)-W3

Tissue 1

Tissue 2

Tissue 3

Liquids volume (μL)

Test Protocol

Start of incubationtime

Tissue 1

Tissue 2

Tissue 3

End of incubationtime

Tissue 1

Tissue 2

Tissue 3










ADDITIONAL CONTROL FOR COLORING TEST SUBSTANCES


Test substanceName or Code

N°……….. N°……….. N°……….. N°……….. N°……….. N°……….. N°………..

Solids Weightbefore application

(mg)W1

Tissue 1

Tissue 2

Tissue 3

Test substanceweight (mg)

W2

Tissue 1

Tissue 2

Tissue 3

Solids weight afterapplication (mg)

W3

Tissue 1

Tissue 2

Tissue 3

Test substanceweight

applied (mg)W=(W1+W2)-W3

Tissue 1

Tissue 2

Tissue 3

Liquids volume (μL)

Test Protocol

Start of incubationtime

Tissue 1

Tissue 2

Tissue 3

End of incubationtime

Tissue 1

Tissue 2

Tissue 3










WEIGHING OF SOLID AND STICKY TEST SUBSTANCES


Code substance N°……….. N°……….. N°……….. N°……….. N°……….. N°……….. N°………..

Solids weighbefore aplication

(mg)

W1

Tissue 1

Tissue 2

Tissue 3

Test substanceweigh (mg)

W2

Tissue 1

Tissue 2

Tissue 3

Solids weigh afterapplication (mg)

W3

Tissue 1

Tissue 2

Tissue 3

Test substanceweigh applied

(mg)

W4=(W1+W2)-W3

Tissue 1

Tissue 2

Tissue 3










TIME PROTOCOLE

Note: Keep 1 minute interval between each tissue application. We recommend keeping some minuteswithout test substance application just before washing in order to be ready in time for this latter


Step DateSet 1 Set 2

RemarksStart Stop Start Stop

Pre-incubation

Application

(42min ± 1min)

Rinsing

Post-incubation

(42h ± 1h)

MTT test

(3h ± 5min)

Extration

(2h ± 10min)

OD reading

(570 ± 30nm)


QC ID: …………….. Date: ……………….. Signature: ……………..







8. BIBLIOGRAPHIC REFERENCES / REPORTSAnon (2003). United Nations (UN). Skin Corrosion/Irritation. UN, Globally harmonized system ofclassification and labelling of chemicals. UN, New York and Geneva, pp. 123-135.

De Brugerolle de Fraisinette A., Picarles V., Chibout S., Kolopp M., Medina J., Burtin P., Ebelin ME.,Osborne S., Mayer F.K., Spake A., Rosdy M., De Wever B., Ettlin RA., and Cordier A. (1999). Predictivityof an in vitro model for acute and chronic skin irritation (SkinEthic®) applied to the testing of topicalvehicles. Cell Biol. Toxicology. 15: 121-135.

Kandárová H., Liebsch M., Schmidt E., Genshow E., Traue D., Meyer K., Spielmann H., Steinoff C.,Tornier C., De Wever B., Rosdy M (2006b). Assessment of the skin irritation potential of chemicals byusing the SkinEthic reconstructed human epidermal model and the common skin irritation protocolevaluated in the ECVAM skin irritation study. Alternatives to Laboratory Animals. 34: 393-406.

OECD (2002): OECD guideline for testing of Chemicals N° 404: Acute Dermal Irritation/Corrosion 6pp.Organization for Economic Cooperation and Development, Paris, France.

OECD (2003): OECD guideline for testing of Chemicals N° 431: In Vitro Skin Corrosion: Human SkinModel Test. Organisation for Economic Cooperation and Development, Paris, France

Prunerias M., Régnier M., Woodley D., (1983). Methods for cultivation of keratinocytes withan air-liquid interface. Journal of Investigative Dermatology. 81(sup n°1): 28s-33s.

Rosdy M., and Clauss LC.(1990) Terminal epidermal differentiation of human keratinocytes grown inchemically defined medium on inert filter substrates at the air-liquid interface. J. Invest. Dermatol. 95:409-414.





skin irritation test for the prediction of acute ......a total of 20 test substances, consisting of...

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